LOCUS       sequence-221968-        5929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a regulatable secretory cargo for yeast at the LEU2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5929)
  AUTHORS   Casler JC, Papanikou E, Barrero JJ, Glick BS
  TITLE     Maturation-driven transport and AP-1-dependent recycling of a 
            secretory cargo in the Golgi.
  JOURNAL   J Cell Biol. 2019 May 6;218(5):1582-1601. doi: 
            10.1083/jcb.201807195. Epub 2019 Mar 11.
  PUBMED    30858194
REFERENCE   2  (bases 1 to 5929)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1083/jcb.201807195"; journalName: "J Cell Biol"; date: 
            "2019-05-6- 6"; volume: "218"; issue: "5"; pages: "1582-1601"
COMMENT     Sequence Label: YIplac128-pOst1-ESCargo
FEATURES             Location/Qualifiers
     source          1..5929
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..40)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     140..159
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        283..713
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             808..1479
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHKA
                     LKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     primer_bind     complement(985..1005)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1203..1222
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1397..1420
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1492..1812
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="GVQVETISPGDGRTFPKRGQTVVVHYTGMLEDGKKLDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGVIPPHATLVFDVELL
                     KLE"
     terminator      1826..2073
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1842..1860)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2092..2108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2092..2108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2105..2127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2116..2132
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2140..2146,2147..2164,2165..2170))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2185..2206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2323..2340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2494..3082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2574..2593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3253..4044,4045..4113))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3876..3895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4114..4218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4286..4304
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4306..4711
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             4712..5806
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
       61 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
      121 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
      181 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcggac cttaatacat
      241 tcagacactt ctgcggtatc accctactta ttcccttcga gattatatct aggaacccat
      301 caggttggtg gaagattacc cgttctaaga cttttcagct tcctctattg atgttacacc
      361 tggacacccc ttttctggca tccagttttt aatcttcagt ggcatgtgag attctccgaa
      421 attaattaaa gcaatcacac aattctctcg gataccacct cggttgaaac tgacaggtgg
      481 tttgttacgc atgctaatgc aaaggagcct atataccttt ggctcggctg ctgtaacagg
      541 gaatataaag ggcagcataa tttaggagtt tagtgaactt gcaacattta ctattttccc
      601 ttcttacgta aatatttttc tttttaattc taaatcaatc tttttcaatt ttttgtttgt
      661 attcttttct tgcttaaatc tataactaca aaaaacacat acataaacta aaagaattca
      721 aaaatgaggc aggtttggtt ctcttggatt gtgggattgt tcctatgttt tttcaacgtg
      781 tcttctgctg cgccggtgaa caccaccgat agcactgaga acgtcatcaa gcccttcatg
      841 cgcttcaagg tgcacatgga gggctcagta aatggtcatg agttcgagat cgagggcgag
      901 ggcgagggca agccctacga gggcacccag accgccaagc tgcaggtgac caagggcggc
      961 cccctgccct tcgcctggga catcctgtcc ccccagttcc agtacggctc caaggtgtac
     1021 gtgaagcacc ccgccgacat ccccgactac aagaagctgt ccttccccga gggcttcaag
     1081 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1141 ctgcaggacg gcaccttcat ctaccacgtg aagttcatcg gcgtgaactt cccctccgac
     1201 ggccccgtaa tgcagaagaa gactctgggc tgggagccct ccaccgagcg cctgtacccc
     1261 cgcgacggcg tgctgaaggg cgagatccac aaggcgctga agctgaaggg cggcggccac
     1321 tacctggtgg agttcaagtc aatctacatg gccaagaagc ccgtgaagct gcccggctac
     1381 tactacgtgg actccaagct ggacatcacc tcccacaacg aggactacac cgtggtggag
     1441 cagtacgagc gcgccgaggc ccgccaccac ctgttccagg gatcctctag aggagtgcag
     1501 gtggaaacca tctccccagg agacgggcgc accttcccca agcgcggcca gaccgttgtg
     1561 gtgcactaca ccgggatgct tgaagatgga aagaaactag attcctcccg ggacagaaac
     1621 aagcccttta agtttatgct aggcaagcag gaggtgatcc gaggctggga agaaggggtt
     1681 gcccagatga gtgtgggtca gagagccaaa ctgactatat ctccagatta tgcctatggt
     1741 gccactgggc acccaggcgt tatcccacca catgccactc tcgtcttcga tgtggagctt
     1801 ctaaaactgg aataaactag agtcatcatg taattagtta tgtcacgctt acattcacgc
     1861 cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
     1921 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
     1981 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
     2041 aaggttttgg gacgctcgaa ggctttaatt tgcaagcttg gcgtaatcat ggtcatagct
     2101 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2161 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2281 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2341 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2401 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2461 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2521 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2581 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2641 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2701 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2761 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2821 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2881 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2941 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3001 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3061 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3121 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3181 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3241 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3301 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3361 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3421 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3481 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3541 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3601 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3661 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3721 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3781 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3841 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3901 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3961 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4021 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4081 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4141 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4201 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     4261 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccaactg tgggaatact
     4321 caggtatcgt aagatgcaag agttcgaatc tcttagcaac cattattttt ttcctcaaca
     4381 taacgagaac acacaggggc gctatcgcac agaatcaaat tcgatgactg gaaatttttt
     4441 gttaatttca gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt gggagaaaaa
     4501 ggaaaggtga gagcgccgga accggctttt catatagaat agagaagcgt tcatgactaa
     4561 atgcttgcat cacaatactt gaagttgaca atattattta aggacctatt gttttttcca
     4621 ataggtggtt agcaatcgtc ttactttcta acttttctta ccttttacat ttcagcaata
     4681 tatatatata tttcaaggat ataccattct aatgtctgcc cctaagaaga tcgtcgtttt
     4741 gccaggtgac cacgttggtc aagaaatcac agccgaagcc attaaggttc ttaaagctat
     4801 ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat catttaattg gtggtgctgc
     4861 tatcgatgct acaggtgttc cacttccaga tgaggcgctg gaagcctcca agaaggctga
     4921 tgccgttttg ttaggtgctg tgggtggtcc taaatggggt acaggtagtg ttagacctga
     4981 acaaggttta ctaaaaatcc gtaaagaact tcaattgtac gccaacttaa gaccatgtaa
     5041 ctttgcatcc gactctcttt tagacttatc tccaatcaag ccacaatttg ctaaaggtac
     5101 tgacttcgtt gttgtcagag aattagtggg aggtatttac tttggtaaga gaaaggaaga
     5161 cgatggtgat ggtgtcgctt gggatagtga acaatacacc gttccagaag tgcaaagaat
     5221 cacaagaatg gccgctttca tggccctaca acatgagcca ccattgccta tttggtcctt
     5281 ggataaagct aatgttttgg cctcttcaag attatggaga aaaactgtgg aggaaaccat
     5341 caagaacgaa tttcctacat tgaaggttca acatcaattg attgattctg ccgccatgat
     5401 cctagttaag aacccaaccc acctaaatgg tattataatc accagcaaca tgtttggtga
     5461 tatcatctcc gatgaagcct ccgttatccc aggttccttg ggtttgttgc catctgcgtc
     5521 cttggcctct ttgccagaca agaacaccgc atttggtttg tacgaaccat gccacggttc
     5581 tgctccagat ttgccaaaga ataaggtcaa ccctatcgcc actatcttgt ctgctgcaat
     5641 gatgttgaaa ttgtcattga acttgcctga agaaggtaag gccattgaag atgcagttaa
     5701 aaaggttttg gatgcaggta tcagaactgg tgatttaggt ggttccaaca gtaccaccga
     5761 agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt gcttaaaaag attctctttt
     5821 tttatgatat ttgtacataa actttataaa tgaaattcat aatagaaacg acacgaaatt
     5881 acaaaatgga atatgttcat agggtaggcc ctttcgtctc gcgcgtttc
//
